Search

The contribution of antigen-driven B-cell adaptive immune responses within the inflamed muscle of inflammatory myopathies (IMs) is largely unknown. In this study, we investigated the immunoglobulin V(H) gene repertoire, somatic hypermutation, clonal diversification, and selection of infiltrating B cells in muscle biopsies from IM patients (dermatomyositis and polymyositis), to determine whether B cells and/or plasma cells contribute to the associated pathologies of these diseases. The data reveal that Ig V(H) gene repertoires of muscle-infiltrating B cells deviate from the normal V(H) gene repertoire in individual patients, and differ between different types of IMs. Analysis of somatic mutations revealed clonal diversification of muscle-infiltrating B cells and evidence for a chronic B-cell response within the inflamed muscle. We conclude that muscle-infiltrating B cells undergo selection, somatic hypermutation and clonal diversification in situ during antigen-driven immune responses in patients with IMs, providing insight into the contribution of B cells to the pathological mechanisms of these disorders., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cmu exons with a set of downstream IgH constant region (C(H)) exons. Individual sets of C(H) exons are flanked upstream by long (1-10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Smu region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region-specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sgamma1 region was replaced with wild-type (WT) or synthetic 2-kb Sgamma3 sequences or a synthetic 2-kb Sgamma1 sequence. We found that both the inserted endogenous and synthetic Sgamma3 sequences functioned similarly to a size-matched synthetic Sgamma1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sgamma3 can function similarly to Sgamma1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype-specific functions of other endogenous S regions.

Survival of patients with B cell chronic lymphocytic leukemia (B-CLL) can be predicted by analysis of mutations in the immunoglobulin heavy chain variable gene (IGHV). Patients without mutations (unmutated [UM]) are at greater risk for disease progression and death than patients with mutations (M). Despite this broad prognostic difference, there remains wide intragroup variation in the clinical outcome of UM patients, especially those with low/intermediate Rai risk disease. We evaluated UM B-CLL patients with low/intermediate Rai risk to determine the relationship between IGHV, IGH diversity (IGHD), and IGH joining (IGHJ) gene usage and time to treatment (TTT). Irrespective of IGHV usage, UM patients whose B-CLL cells expressed the IGHD3-3 gene had a significantly shorter TTT than other UM B-CLL patients, and specifically, use of the IGHD3-3 gene in reading frame 2 (RF2) predicted shorter TTT. As expected, Rai risk was the best single prognostic factor for TTT; however, IGHD usage was also a significant variable for TTT. Therefore, both IGHD gene and IGHD RF usage have prognostic relevance in UM B-CLL patients with low/intermediate Rai risk disease. In addition, these data support the concept that antigen-driven selection of specific Ig receptors plays a role in the clinical course of B-CLL.

The more distal enhancers of the immunoglobulin heavy-chain 3' regulatory region, hs3b and hs4, were recently demonstrated as master control elements of germline transcription and class switch recombination to most immunoglobulin constant genes. In addition, they were shown to enhance the accumulation of somatic mutations on linked transgenes. Since somatic hypermutation and class switch recombination are tightly linked processes, their common dependency on the endogenous locus 3' enhancers could be an attractive hypothesis. VDJ structure and somatic hypermutation were analyzed in B cells from mice carrying either a heterozygous or a homozygous deletion of these enhancers. We find that hs3b and hs4 are dispensable both for VDJ assembly and for the occurrence of mutations at a physiologic frequency in the endogenous locus. In addition, we show that cells functionally expressing the immunoglobulin M (IgM) class B-cell receptor encoded by an hs3b/hs4-deficient locus were fully able to enter germinal centers, undergo affinity maturation, and yield specific antibody responses in homozygous mutant mice, where IgG1 antibodies compensated for the defect in other IgG isotypes. By contrast, analysis of Peyer patches from heterozygous animals showed that peanut agglutinin (PNAhigh) B cells functionally expressing the hs3b/hs4-deficient allele were dramatically outclassed by B cells expressing the wild-type locus and normally switching to IgA. This study thus also highlights the role of germinal centers in the competition between B cells for affinity maturation and suggests that membrane IgA may promote recruitment in an activated B-cell compartment, or proliferation of activated B cells, more efficiently than IgM in Peyer patches.

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.

Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen., (Copyright 1999 Academic Press.)

Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e. g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29 mu), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of S mu-S alpha recombination in the plasmid. Recombination of the plasmid in I.29 mu cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29 mu cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.

Rheumatoid factor (RF) is the most common autoantibody found in patients with Sjögren's syndrome (SS). To study the genetic origin and the mechanisms acting behind its generation we have characterized and sequenced the immunoglobulin VH genes used by 10 IgM RF MoAbs derived from peripheral blood of six female patients with pSS. We compared the structure of the RF immunoglobulin VH genes with those obtained previously from rheumatoid arthritis (RA) patients and healthy immunized donors (HID). VH1 and VH4 were each used by four RF clones, one clone was encoded by VH3 family gene and one by VH2 family gene. This distribution frequency was different from that observed in RA, where VH3 was the dominant family, followed by VH1. Eight different germ-line (GL) genes encoded the clones and all of these genes were seen previously in RA and/or HID RF. Five clones rearranged to JH6, four rearranged to JH4 and one to JH5, in contrast to RF from RA and HID, where JH4 was most frequently used. D segment use and CDR3 structure were diverse. Interestingly, three out of four VH4 clones used the GL gene DP-79 that was seen frequently in RA RF. The degree of somatic mutation in the pSS RF was very much lower than seen in RA and HID RF. All the pSS RF clones except three were in or very close to GL configuration. This indicates that there is little role for somatic hypermutation and a germinal centre reaction in the generation of RF from peripheral blood in pSS.

Somatic hypermutation affects Ig genes during T-dependent B cell responses and is characterized by a high frequency of single base substitutions. Hypermutation is not a completely random process; a study of mutations in different systems has revealed the presence of sequence motifs that target mutation. In a recent analysis of the sequences surrounding individual mutated bases in out-of-frame human IgVH genes, we found that the target motifs around mutated G's and C's are reverse complements of each other. This finding suggests that hypermutation acts on both strands of DNA, which contradicts evidence of a strand-dependent mechanism as suggested by an observed bias in A and T mutations and the involvement of transcriptional machinery. We have now extended our database of out-of-frame genes and determined the sequence motifs flanking mutated A and T nucleotides. In addition, we have analyzed the flanking sequences for different types of nucleotide substitutions separately. Our results confirm the relationship between the motifs for G and C mutations and show that the motifs surrounding mutated A's and T's are weaker and do not have the same relationship. Taken together with our observation of A/T strand bias in out-of-frame genes, this observation suggests that there is a semitargeted G/C mutator that is strand-independent and a separate A/T mutator that is strand-dependent and is less reliant on the local target sequence.

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.

A panel of mutant antibodies of the phosphocholine (PC)-binding antibody, T15, was tested for binding to PC-protein, Streptococcus pneumoniae, Trichinella spiralis and Ascaris suum. Relative to wildtype T15, all the mutant antibodies showed differential recognition of the panel of PC-associated antigens. These mutant antibodies contain amino acid replacements in the CDR2 region of the heavy chain variable region, indicating the importance of CDR2 in recognition of carrier determinants. A model of T15 is shown that illustrates the strategic placement of mutations that could allow interaction with determinants associated with PC. A direct implication of this finding is that the T15 antibody combining site accommodates structures larger than phosphocholine and that recognition of associated carrier determinants could be a significant force in shaping the immune response to PC-containing pathogens.

Naive B cells expressing IgM and IgD on their surface have no or little somatic mutations in V genes. We have demonstrated that the human IgM+IgD+B cell clone (0-81), which expresses nephritogenic idiotypes, produces IgM anti-DNA antibodies which show monospecificity to DNA. Using a DNA probe which specifically links to the VH gene of antibody 0-81, we identified the counterpart germ-line V gene of 0-81, V3-7, which appears to be used by pathogenic autoantibodies in humans. Clone 0-81, which may belong to naive B cells in terms of cell phenotype, uses a somatically mutated V3-7 gene. We further studied DNA sequences of V3-7 genes in circulating IgM+IgD+B cells from normal subjects and patients with systemic lupus erythematosus (SLE). The results revealed that rearranged V3-7 genes in IgM+IgD+B cells from patients with SLE contained somatically mutated sequences at significantly increased frequencies. These data indicate an abnormal maturation of B cells in autoimmune states that may be associated with an escape of self-reactive B cells from the elimination process in the germinal center.

To investigate the origin of disease-associated IgM autoantibodies (AAb), we compared the genetic and structural characteristics of IgM AAb from autoimmune prone motheaten (mev) mice with natural autoantibodies (NAAb) from normal background C57/BL6 strain. Six hybridoma-derived IgM molecules each were obtained both from mev mice, at the terminal stage of systemic autoimmune disease, and from mitogen-stimulated C57/BL6 mice. These were randomly selected for VH J558 gene expression (aberrantly expressed in mev mice). The variable regions of the IgM molecules, both from autoimmune and normal mice, were encoded by unmutated germline VH genes. Disease-associated AAb from mev mice were predominantly encoded by the J558 subfamily 186.2, whereas five J558 subfamilies were utilized in NAAb originating from normal mice. Junctional diversity as a result of N or P nucleotide insertions and D-D fusions was noted among IgMs originating from both mev (mostly B-1 lymphocytes) and C57BL/6 (mostly B-2 lymphocytes) mice. Interestingly, all six J558+ IgMs from mev mice showed a restricted CDR3 length of 10 amino acids, with similar hydrophobicity indices. Four unique V-D-J rearrangements were observed among these IgMs. None of the IgMs were polyreactive and three of the six were subsequently observed to express monospecific autoreactivity with synthetic peptides (residues 81-92 and 37-53) representing segments of the T cell CD4-accessory molecule. Three IgM antibodies had hydrophilic arginine residues in their CDR3 heavy chain region. By contrast, all six J558+ IgMs from C57/BL6 mice had variable CDR3 length, distinct VDJ rearrangements and a local negative charge in the CDR3 region. Four of these IgMs demonstrated polyreactivity with multiple conserved autoantigens and, hence, were classified as NAAb. These findings provide evidence for either positive or impaired negative selection of B-1 lymphocytes secreting disease-associated IgM AAb in mev mice. This likely results from a reduced threshold of responsiveness to autoantigens due to PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size and hydrophobicity pattern of the CDR3 of the heavy chain allow structural distinction between monospecific disease-associated IgM AAb and the polyreactive IgM NAAb.

Previous work on the cis-acting elements that control heavy chain variable region (VH) gene somatic hypermutation has indicated the presence of an as yet unidentified element(s) 3' of the intron enhancer that is necessary for high rate mutation. Examination of cis-acting elements involved in kappa light chain V gene hypermutation has demonstrated a requirement for both the intronic and 3' kappa enhancers in this process. To examine whether the 3'alpha heavy chain enhancer [3'alpha E(hs1,2)] is required for somatic hypermutation of VH genes, we generated two types of transgenic mice. One type was generated using a construct containing a VH promoter, a rearranged VDJ, the heavy chain intronic enhancer, and the murine heavy chain 3'alpha E(hs1,2). The transgenes in the second lines were similar to the transgenes in the first with the addition of a second complete matrix attachment region (MAR) 3' of the heavy chain intronic enhancer, and splice acceptor and polyadenylation sites between the two enhancers. Analysis of both transgenes revealed levels of mutation at least 10-fold lower than endogenous VH genes. These data suggest that the 3'alpha E(hs1,2) does not play a role analogous to the 3' kappa enhancer in the regulation of the hypermutation process. Moreover, in one of the transgenes, the presence of the 3'alpha E(hs1,2) resulted in a lack of transcription in vivo, suggesting a negative regulatory role for this enhancer in certain contexts.

Previous experiments have suggested an important role for the Ig enhancers and transcription in targeting somatic hypermutation. To determine whether the requirement of the enhancers is Ig chain specific, we analyzed two lambda 2 light chain transgenes under the control of different enhancers, either the lambda 2-4 enhancer or the heavy chain intron enhancer. The transgenes were amplified and cloned from B220+PNAhigh B cells from either Peyer's patches (PP) or SRBC-immunized spleen. The lambda 2 transgene under the control of the lambda 2-4 enhancer underwent mutation in both the PP and splenic B cell populations, but at a frequency lower than endogenous light chain genes. This requirement for the lambda 2-4 enhancer could be replaced by the heavy chain intron enhancer. Interestingly, the heavy chain intron enhancer-driven lambda 2 construct showed evidence of statistically significant mutation in the PP B cell population, but not the spleen-derived population. This difference in somatic hypermutation suggests that a lower mutation frequency can be more readily detected in B220+PNAhigh B cells isolated from the PP. The ability of the heavy chain intron enhancer to replace the lambda 2-4 enhancer shows that the requirement for the Ig enhancers in somatic hypermutation is not Ig locus specific. Given that these Ig enhancers have very few elements in common, our results further suggest that the Ig enhancers are primarily important in the timing of transcription in the mutating B cell population.

We recently described mutants of the murine anti-phosphocholine Ab T15, with changes in heavy chain complementarity determining region 2 (HCDR2) that caused loss of secretion. Surprisingly, the T15 HCDR2 mutations did not alter secretion when placed into the related anti-phosphocholine Ab D16, which differs from T15 only in HCDR3 and light (L) chain. Here, we exploit the differences between these two Abs to assess the basis of the secretion defect. The T15 L chain is not secreted in the absence of heavy (H) chain. In contrast, D16 L chain is secreted in the absence of H chain, as are most L chains. We co-expressed the T15 wild-type (wt) and mutant H chains with the D16 L chain, as well as with another secreted L chain, J558L. The mutant H chains were not secreted when expressed with either heterologous L chain. These results establish that the T15 L chain is not uniquely associated with the defect. The T15 and D16 Abs also differ in HCDR3 length in that D16 lacks four amino acid residues (Ser99, Ser100, Tyr100a, Trp100b) present in T15. We deleted these four residues from T15 wt and mutant H chains. Secretion of T15 wt was unaffected by the deletion, but shortening HCDR3 restored secretion in the HCDR2 mutants regardless of L chain association. Together these data demonstrate that both the HCDR2 and HCDR3 domains contain structural information that may affect the secretion competence of Abs.

Early IgM+ B cells express little or no membrane IgD due to the low abundance of delta mRNA. Extensive transcriptional termination regulated by sequences in the intronic region between mu and delta heavy chain genes may be the primary reason for the lack of delta gene transcription. We have examined the effect of deletion of these sequences on the regulation of IgM and IgD heavy chain gene expression in transfectants as well as mice carrying this otherwise intact transgene. By run-on transcriptional measurement, we show that the delta exons are transcribed in bone marrow B cells from these transgenic mice. However, in spite of the induced premature synthesis of the full-length mu-delta transcript in pre-B cells, processing to delta mRNA does not occur until the lymphocytes express cell surface IgM. Therefore, during B cell development, synthesis of the full-length transcript is a necessary but not sufficient condition for initiation of delta mRNA synthesis. Furthermore, unexpectedly, the abrogation of transcriptional termination was found to also affect the processing of the primary transcript to microM mRNA. These results show that expression of IgD in primary B cells is stringently regulated and closely linked to IgM expression.

Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis-acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti-NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5' IgH sequences, a rearranged VDJ region, the 5' matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3' IgH sequences (an alpha constant region and the 3' enhancer) and either the original VDJ segment or a substituted T cell receptor beta segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3' enhancer.

The normal counterpart of the neoplastic B cells occurring in Burkitt's lymphomas (BL) is an issue of controversial debate. To clarify this matter, a semi-nested primer polymerase chain reaction was performed to amplify the VDJ rearrangements of the immunoglobulin heavy chain (VH) gene of DNA extracts from 10 (8 sporadic and 2 endemic) BL cases. The resulting amplificates were sequenced for comparison with known germ line VH segments. The control cases comprised six cases of B cell chronic lymphocytic leukemia and six cases of mantle cell lymphoma known to display naive nonmutated, ie, pre-germinal center VH configurations; and eight cases of follicular center lymphoma known to display mutated VH genes with signs of a still-ongoing mutation reaction, characteristic for germinal center cells and lymphomas that derive therefrom. The results of this approach revealed that both sporadic and endemic BL express mutated VH genes with a mutation frequency considerably lower (4.9% and 5.4%, respectively) than that observed in follicular center lymphoma (11.8%). In addition, after subcloning the amplificates, sequence analysis revealed no signs of ongoing mutations. These results led us to conclude that the derivation of neoplastic B cells in BL is definitely not from naive, nonmutated pre-germinal center B cells. Instead, our findings support the view that BL cells stem either from early centroblasts that are arrested after an initial hypermutation reaction, or from germinal center B cells that have differentiated in terms of surface immunoglobulin profile and mutation pattern but not in terms of morphology and proliferation toward SIgM+ IgD- memory B cells because of the deregulated c-myc gene expression.

We have evaluated the importance of the OCTA site in both the promoter and enhancer regions for the induction of enhancement of IgH chain gene transcription after B cell activation. These studies show that although occupancy of the OCTA site in the promoter is critical for basal transcription of the mu gene, it is not necessary for the increase in transcription induced by in vivo activation. On the other hand, the OCTA site in the enhancer is necessary for neither basal transcription nor in vivo activation of transcription; however, occupancy of this site is required for further up-regulation in transcription of the mu gene in pre-activated cells. These results indicate that different mechanisms may be involved in the activation of resting versus in vivo stimulated B lymphocytes. The findings are discussed in relation to the phenotype described for Oct-2-deficient mice.

Murine B cells proliferating in the germinal centers of peripheral lymphoid tissue accumulate mutations in their rearranged variable regions, a diversification process which contributes to affinity maturation of the antibody response. The highly targeted nature of the hypermutation process could be explained by a somatic gene conversion mechanism. Well characterized examples of such an activity in B cells are seen during diversification of the chicken and rabbit Ig repertoires. The genomic organization, low complexity and high degree of homology exhibited by the four members of the murine VHS107 gene family suggested that these gene segments may be suitable candidates for the search of gene conversion events derived from upstream VHS107 counterparts. After an immune response to a complex T cell-dependent antigen (sheep red blood cells), rearranged V13, V11 and V1 genes were isolated from splenic extrafollicular and germinal center B cells. Extensive somatic mutation was evident in V11 and V1 sequences. When these sequences were examined, as well as V1 sequences isolated from phosphorylcholine-specific hybridomas, the observed nucleotide changes were not associated with any gene conversion between family members, suggesting instead that they arose by a mechanism which introduces point mutations.

Following infection with EBV, patients have selectively raised serum levels of immunoglobulins encoded by the VH4-21 gene. In order to follow the detailed pattern of usage of the VH4-21 gene by blood B lymphocytes of a typical patient during infection, EBV lines were established, and transformed B cells were hybridized and cloned. In addition, to widen the genetic analysis, cDNA preparations from the EBV transformants using the gene were also analysed by polymerase chain reaction, cloning and sequencing. The majority (12/15) of the clonally distinct sequences derived from IgM utilized the VH4-21 gene in germ line configuration; however, 3/15 showed replacement mutations. For one of these, a heterogeneous pattern of mutation within the clone indicated ongoing mutation, and one sequence contained a stop codon. Three distinct clones which had rearranged to C gamma were obtained, and all were extensively mutated, with some evidence for a role for antigen in selection. Following resolution of the infection, no VH4-21-encoded products were detectable by this approach. It appears therefore that infection with EBV leads to selective activation, mutation and class switching of the VH4-21 gene, with the unusual feature that B cells harbouring deleterious mutations in the functional gene are able to survive in the circulation.